Serine proteases they comprise a diverse class of enzymes having a wide range of specificities and biological functions. Stroud, R. Sci. Amer., 131:74–85. Despite their functional diversity, the catalytic machinery of serine proteases has been approached by at least two genetically distinct families of enzymes: 1) the subtilisins and 2) the mammalian chymotrypsin-related and homologous bacterial serine proteases (e.g., trypsin and S. gresius trypsin). These two families of serine proteases show remarkably similar mechanisms of catalysis. Kraut. J. (1977), Annu. Rev. Biochem, 46:331–358. Furthermore, although the primary structure is unrelated, the tertiary structure of these two enzyme families brings together a conserved catalytic triad of amino acids consisting of serine, histidine and aspartate.
Subtilisins are serine proteases (approx. MW 27,500) which are secreted in large amounts from a wide variety of Bacillus species and other microorganisms. The protein sequence of subtilisin has been determined from at least nine different species of Bacillus. Markland, F. S., et al. (1983), Hoppe-Seyler's Z. Physiol. Chem., 364:1537–1540. The three-dimensional crystallographic structure of subtilisins from Bacillus amyloliquefaciens, Bacillus licheniformis and several natural variants of B. lentus have been reported. These studies indicate that although subtilisin is genetically unrelated to the mammalian serine proteases, it has a similar active site structure. The x-ray crystal structures of subtilisin containing covalently bound peptide inhibitors (Robertus, J. D. et al. (1972), Biochemistry, 11:2439–2449) or product complexes (POULOS, et al. (1976), J. Biol. Chem., 251:1097–1103) have also provided information regarding the active site and putative substrate binding cleft of subtilisin. In addition, a large number of kinetic and chemical modification studies have been reported for subtilisin; Svendsen, B. (1976), Carlsberg Res. Commun., 41:237–291; Markland, F. S. Id.) as well as at least one report wherein the side chain of methionine at residue 222 of subtilisin was converted by hydrogen peroxide to methionine-sulfoxide (Stauffer, D. C., et al. (1965), J. Biol. Chem., 244:5333–5338) and extensive site-specific mutagenesis has been carried out (Wells and Estell (1988) TIBS 13:291–297)
A common issue in the development of a protease variant for use in a detergent formulation is the variety of wash conditions including varying detergent formulations that a protease variant might be used in. For example, detergent formulations used in different areas have different concentrations of their relevant components present in the wash water. For example, a European detergent system typically has about 4500–5000 ppm of detergent components in the wash water while a Japanese detergent system typically has approximately 667 ppm of detergent components in the wash water. In North America, particularly the United States, a detergent system typically has about 975 ppm of detergent components present in the wash water. Surprisingly, a method for the rational design of a protease variant for use in a low detergent concentration system, a high detergent concentration system, and/or a medium detergent concentration system as well as for use in all three types of detergent concentration systems has been developed.